This proposed multidisciplinary project will utilize electrophysiological, pharmacological and biochemical techniques to elucidate the mechanisms whereby (-)-delta9-tetrahydrocannabinol (THC) and other cannabimimetic agents depress the function of the voltage-gated sodium channel in N1E-115 neuroblastoma cells. This will be done by testing our working hypothesis that (a) depression of the sodium current occurs as a consequence of non- receptor-mediated actions of the cannabinoids, (b) the cannabinoids directly or indirectly stimulate the activity of protein kinase C, and (c) activation of protein kinase C causes phosphorylation of the sodium channel and a consequent reduction in peak sodium current. The cannabinoids might stimulate protein kinase C by a direct action on the enzyme; alternatively, they might increase the intracellular concentration of arachidonic acid, a cis-unsaturated fatty acid which is known to stimulate protein kinase C. Experiments will seek to determine how the THC-induced increase in intracellular arachidonic acid takes place and its relationship to the reduction of sodium current. Conventional and nystatin-permeabilized whole-cell voltage clamp techniques will be used to quantify sodium current in most experiments. Cannabimimetic agents to be studied in addition to THC include (a) (+)- delta9-tetrahydrocannabinol, the inactive stereoisomer of THC, (b) anandamide, the putative natural ligand for the THC receptor, (c) metabolism-resistant congeners of anandamide, and (d) cannabidiol, a cannabinoid which lacks psychoactivity but depresses the neuronal action potential. Single sodium channels will be studied in cell-attached membrane patches in order to determine how THC and cannabimimetic agents reduce the whole- cell peak sodium current by altering channel opening and closing. In particular, the effect of THC treatment on the average channel current, the probability of channel opening, and the number of available sodium channels will be examined. In addition, studies of single channels in inside-out isolated membrane patches will be used to investigate the actions of THC and arachidonic acid on the sodium channel in the presence and absence of exogenously supplied protein kinase C and will determine which specific isoforms of PKC are involved in modulating channel function. If, as is hypothesized, cannabimimetic agents directly or indirectly affect protein kinase C, then these agents could potentially alter signaling within the nervous system, neurotransmitter release, long-term potentiation, several diseases of the nervous system, and the functioning of the signal transduction system in general.